Phospholipids of ethambutol-susceptible and resistant strains ofMycobacterium smegmatis

The composition, subcellular distribution and rate of synthesis of phospholipids were compared in ethambutol susceptible and resistant strains ofMycobacterium smegmatis. Significant quantitative alterations in phospholipids accompanied the acquisition of resistance, whereas fatty acyl group composition of total phospholipid remained the same in ethambutol resistant and susceptible strains. Cell wall of resistant strain exhibited an accumulation of phospholipids and a decrease in the degree of unsaturation of phospholipid fatty acyl groups. Changes in the cell wall phospholipid composition may contribute to resistance ofMycobacterium smegmatis to ethambutol.     

Cell wall composition of ethambutol susceptible and resistant strains of Mycobacterium smegmatis ATCC 607

Changes in the cell wall that accompany acquisition of ethambutol (EMB) resistance ina single step mutant of Mycobacterium smegmatis ATCC 607 were analysed. Quantitative changes were seen in the chemical constituents of the cell wall of resistant cultures in comparison with EMB-susceptible M. smegmatis . Alterations in the binding of 1-anilinonaphthalene-8-sulphonate (ANS) were suggestive of structural changes in the cell surface.     

脓肿分支枝杆菌embB基因耐乙胺丁醇的分析

目的 分析脓肿分支枝杆菌的embB基因,以探讨其耐乙胺丁醇的分子机制.方法 用16S rRNA基因序列分析法鉴定5株脓肿分枝杆菌临床株,测定乙胺丁醇对临床株及标准株( ATCC 19977)的最低抑菌浓度(MIC).PCR扩增embB基因的全序列,将所测序列进行生物信息学分析.结果 乙胺丁醇对5株脓肿分支杆菌标准株和临床株的MIC均为128μg/mL.,属高度耐药.从脓肿分支枝杆菌的标准株和临床株均扩增出约3200 bp片段,与GenBank中脓肿分支枝杆菌标准株的embB基因大小一致.5株临床株与标准株比较,其核苷酸序列存在9个点突变,在突变位点所编码的氨基酸序列中,仅第18位、87位、770位密码子编码与标准株不同的氨基酸.6株脓肿分支枝杆菌的embB基因与对EMB敏感的结核分支杆菌标准株(H37RV)的相应基因序列比较,第303-305位密码子的核苷酸序列存在差异,但仅第303、304位核苷酸编码的氨基酸序列不同,第306位密码子的核苷酸序列无差异.结论 脓肿分支杆菌对乙胺丁醇的耐药并非embB基因的突变所致,为embB基因天然存在结构的不同,属于天然耐药.结构的差异与第306位密码子无关,可能与第303、304密码子有关.     

Biochemical mechanism of combined effect of ethambutol and sparfloxacin against Mycobacterium smegmatis

M. smegmatis cells grown in the presence of combination of ethambutol (EMB) and sparfloxacin (SPX) had decreased level of total cellular lipids as compared to control as well as cells grown in the presence of sub-inhibitory concentration (MIC50) of individual drugs. Amongst various phospholipids analyzed, maximum decrease was observed in the content of phosphatidylinositolmannosides (PIMs) of the cells grown in combination of EMB and SPX. In contrast, the subcellular distribution of phospholipids revealed a significant increase in PIMs content of both cell wall and cell membrane of the cells grown in the presence of combination of drugs as compared to control as well as individual drugs. Mycolic acids of M. smegmatis cells were found to be main targets as combination of drugs resulted in significant decrease in total cellular as well as cell wall mycolic acids as compared to control and individual drugs. Changed lipid composition of M. smegmatis cells grown in the presence of MIC50 of EMB, SPX and combination resulted in significant surface changes as was evident from decreased limiting fluorescence (Fmax) intensity of 1-anilinonaphthalene-8-sulfonate (ANS). Thus, the results of this study suggested that ethambutol and sparfloxacin in combination exerted their antimycobacterial effect principally due to their action on phosphatidylinositolmannosides (PIMs) and mycolic acids, which form the permeability barrier of mycobacteria.     

Phospholipid biosynthesis and solvent tolerance in Pseudomonas putida strains.

The role of the cell envelope in the solvent tolerance mechanisms of Pseudomonas putida was investigated. The responses of a solvent-tolerant strain, P. putida Idaho, and a solvent-sensitive strain, P. putida MW1200, were examined in terms of phospholipid content and composition and of phospholipid biosynthetic rate following exposure to a nonmetabolizable solvent, o-xylene. Following o-xylene exposure, P. putida MW1200 exhibited a decrease in total phospholipid content. In contrast, P. putida Idaho demonstrated an increase in phospholipid content 1 to 6 h after exposure. Analysis of phospholipid biosynthesis showed P. putida Idaho to have a higher basal rate of phospholipid synthesis than MW1200. This rate increased significantly following exposure to xylene. Both strains showed little significant turnover of phospholipid in the absence of xylene. In the presence of xylene, both strains showed increased phospholipid turnover. The rate of turnover was significantly greater in P. putida Idaho than in P. putida MW1200. These results suggest that P. putida Idaho has a greater ability than the solvent-sensitive strain MW1200 to repair damaged membranes through efficient turnover and increased phospholipid biosynthesis.     

Alterations in the phospholipid composition of Rhodopseudomonas sphaeroides and other bacteria induced by Tris.

Alterations in the phospholipid head group composition of most strains of Rhodopseudomonas sphaeroides, as well as Rhodopseudomonas capsulata and Paracoccus denitrificans, occurred when cells were grown in medium supplemented with Tris. Growth of R. sphaeroides M29-5 in Tris-supplemented medium resulted in the accumulation of N-acylphosphatidylserine (NAPS) to as much as 40% of the total whole-cell phospholipid, whereas NAPS represented approximately 28 an 33% of the total phospholipid when R. capsulata and P. denitrificans respectively, were grown in medium containing 20 mM Tris. The accumulation of NAPS occurred primarily at the expense of phosphatidylethanolamine in both whole cells and isolated membranes of R. sphaeroides and had no detectable effect on cell growth under either chemoheterotrophic or photoheterotrophic conditions. Yeast extract (0.1%) and Casamino Acids (1.0%) were found to be antagonistic to the Tris-induced (20 mM) alteration in the phospholipid composition of R. sphaeroides. The wild-type strains R. sphaeroides 2.4.1 and RS2 showed no alteration in their phospholipid composition when they were grown in medium supplemented with Tris. In all strains of Rhodospirillaceae tested, as well as in P. denitrificans, NAPS represented between 1.0 and 2.0% of the total phospholipid when cells were grown in the absence of Tris. [32P]orthophosphoric acid entered NAPS rapidly in strains of R. sphaeroides that do (strain M29-5) and do not (strain 2.4.1) accumulate this phospholipid in response to Tris. Our data indicate that the phospholipid head group composition of many Rhodospirillaceae strains, as well as P. denitrificans, is easily manipulated; thus, these bacteria may provide good model systems for studying the effects of these modifications on membrane structure and function in a relatively unperturbed physiological system.     

Phospholipid composition of Mycobacterium smegmatis susceptible and resistant to antitubercular drugs

The phospholipid composition, distribution and metabolism in mono drug resistant mutants towards antitubercular drugs, viz, streptomycin, ethambutol and isoniazid, were investigated. Though their total phospholipid content was not altered significantly, changes were observed in their individual phospholipid content. Reduced biosynthesis and degradation of phospholipids (monitored by pulse and chase technique using [32P]orthophosphoric acid as a precursor) was observed in all the mutants studied. The subcellular distribution of phospholipids revealed accumulation of phospholipids in the cell walls and reduction in cell membranes of the drug-resistant mutants. Similar alterations were seen in individual phospholipids of these subcellular fractions.     

Membrane phospholipid composition of Escherichia coli affects secretion of periplasmic alkaline phosphatase into the medium

Secretion of alkaline phosphatase (PhoA) encoded by a gene constituent of plasmids has been studied in Escherichia coli strains with controlled synthesis of anionic phospholipids (phosphatidylglycerol and cardiolipin, strain HDL11) and zwitterionic phospholipid (phosphatidylethanolamine, strain AD93). Changing the phospholipid composition of the membrane of these strains leads to an increase in secretion of PhoA, which is usually localized in the periplasm, into the culture medium. This correlates with a higher secretion of exopolysaccharides and lower content of lipopolysaccharide in the outer membrane. The results show the possibility of coupling protein secretion into the medium with biogenesis of cell envelope components in which phospholipids are involved.     

Biochemical studies with bi-resistant mutants (ethambutol plus streptomycin and isoniazid plus streptomycin) ofMycobacterium smegmatis ATCC 607

Biochemical characteristics of bi-resistant mutants (resistant to ethambutol plus streptomycin or isoniazid plus streptomycin) of mycobacteria isolated by replica plating fromMycobacterium smegmatis ATCC were compared with those of the drug-susceptible strains. Reduced incorporation of [¹⁴C]uracil, [³H]lysine and [¹⁴C]acetate into RNA, protein and phospholipids respectively was seen in the resistant mutants. Total phosphorlipids were enhanced in ethambutol plus streptomycin resistant mutant and decreased in isoniazid plus streptomycin resistant mutant. There were similar changes in levels of individual phospholipids. The resistant mutants revealed an accumulation of phospholipids in the cell wall, and a marked decrease of phospholipids in the cell membrane in comparison to the susceptible strain. Several qualitative alterations in the polypeptide profile (with respect to number and molecular weight) of the crude protein extract and of different subcellular compartments were seen in the resistant mutants.     

Selective expression of light-harvesting complexes alters phospholipid composition in the intracytoplasmic membrane and core complex of purple phototrophic bacteria

Phospholipid–protein interactions play important roles in regulating the function and morphology of photosynthetic membranes in purple phototrophic bacteria. Here, we characterize the phospholipid composition of intracytoplasmic membrane (ICM) from Rhodobacter (Rba.) sphaeroides that has been genetically altered to selectively express light-harvesting (LH) complexes. In the mutant strain (DP2) that lacks a peripheral light-harvesting (LH2) complex, the phospholipid composition was significantly different from that of the wild-type strain; strain DP2 showed a marked decrease in phosphatidylglycerol (PG) and large increases in cardiolipin (CL) and phosphatidylcholine (PC) indicating preferential interactions between the complexes and specific phospholipids. Substitution of the core light-harvesting (LH1) complex of Rba. sphaeroides strain DP2 with that from the purple sulfur bacterium Thermochromatium tepidum further altered the phospholipid composition, with substantial increases in PG and PE and decreases in CL and PC, indicating that the phospholipids incorporated into the ICM depend on the nature of the LH1 complex expressed. Purified LH1–reaction center core complexes (LH1–RC) from the selectively expressing strains also contained different phospholipid compositions than did core complexes from their corresponding wild-type strains, suggesting different patterns of phospholipid association between the selectively expressed LH1–RC complexes and those purified from native strains. Effects of carotenoids on the phospholipid composition were also investigated using carotenoid-suppressed cells and carotenoid-deficient species. The findings are discussed in relation to ICM morphology and specific LH complex–phospholipid interactions.     

In vitro synergistic activity between 8-methoxypsoralen and ethambutol, isoniazid, and rifampin when used in combination against Mycobacterium tuberculosis

8-Methoxypsoralen (8-MOP), a naturally occurring furocoumarin found in many plant species, has been reported to have antimycobacterial activity against Mycobacterium tuberculosis strain H37Rv (ATCC 27294). In the present study, we further test the in vitro synergistic activity of 8-MOP and ethambutol (EMB), isoniazid (INH), or rifampin (RMP) against M. tuberculosis. This study showed that 8-MOP has antimycobacterial activity against two drug-sensitive and six drug-resistant clinical isolates of M. tuberculosis, with the minimum inhibitory concentrations of 100–200 and 200–400 μg/mL, respectively. A synergistic antimycobacterial effect between 8-MOP and EMB, INH, or RMP against six drug-resistant strains was observed, with the fractional inhibitory concentration indices (FICIs) of 0.093–0.156, 0.138–0.285 and 0.093–0.262, respectively. The combination of 8-MOP/EMB, 8-MOP/INH, and 8-MOP/RMP displayed either synergistic activity or had no interaction when tested against the two clinical drug-sensitive strains and the standard strain. No antagonism was observed for any drug combination against any of the strains tested. To our knowledge, this is first report that 8-MOP has synergistic activity with first-line antimycobacterial agents.     

Fluorescent phospholipid analogs as microscopic probes for detection of the mycolic acid-containing layer in Corynebacterium glutamicum: detecting alterations in the mycolic acid-containing layer following ethambutol treatment

Corynebacterium glutamicum belongs to the mycolic acid-containing actinomycetes, which also include Mycobacterium, Nocardia, and Rhodococcus. The cells of this group possess a cell wall with a thick outer layer composed primarily of mycolic acid, which functions as a permeability barrier. To investigate the mechanism of mycolic acid-containing layer (mycolate layer) formation, we have developed a fluorescence microscopic technique detecting the mycolate layer in situ. The staining specificity of fluorescence-labeled phospholipid analogs was determined by simultaneous staining with the hydrophobic fluorescent dye Nile Red and peptidoglycan-staining fluorescence-conjugated vancomycin. We found that fluorescence-labeled phospholipid analogs preferentially stain the mycolate layer. Using this technique, we observed the effect of the anti-mycobacterial drug ethambutol on C. glutamicum mycolate-layer formation. Ethambutol interfered specifically with mycolate-layer formation on the division planes and cell poles, while the side-wall mycolate layer was not severely affected. This indicates that mycolate-layer formation occurs mainly on division planes and cell poles in C. glutamicum, where the peptidoglycan layer is actively synthesized.     

结核分枝杆菌耐链霉素和乙胺丁醇分子机制的研究

目的:研究结核分枝杆菌耐链霉素和乙胺丁醇的rpsL和emb B基因突变情况,探讨耐药基因突变与耐药性的关系。方法:通过传统药敏实验和聚合酶链反应(PCR)--单链构象多态性(SSCP)技术初步鉴定62株临床分离株的药敏和rps L、emb B基因。结果:与结核菌标准株H37Rv对照,分析30例TB菌耐链霉素(SM)的rps L基因,发现其突变率为70.0%(21/30),分析29例耐乙胺丁醇(EMB)的emb B基因,该基因的突变率为65.5%(19/29)。结论:部分结核分枝杆菌耐SM和EMB是由于其rps L、emb B基因突变所致,PCR-SSCP银染技术可能成为测定部分结核分枝杆菌耐药的简便、快速的方法。     

Effect of choline on the morphology, growth and phospholipid composition of Fusarium graminearum

Studies were made of the growth kinetics, morphology and phospholipid composition of two strains of Fusarium graminearum, a wild-type strain (A3/5) and a highly branched variant (C106) which arose spontaneously during cultivation of A3/5. No significant difference was observed between the hyphal diameters of the two strains and therefore increased branching of C106 could not be explained in the terms of an increase in hyphal radius in the absence of a change in hyphal growth unit volume. The two strains had the same specific growth rate in batch culture and this was not affected by the addition of up to 1.5 mM-choline to the medium. However, choline increased the mean hyphal extension rate and colony radial growth rate of both strains and this response was correlated with the formation of mycelia which were more sparsely branched than mycelia grown on medium lacking choline. Addition of betaine, choline, ethanolamine, monomethylethanolamine or dimethylethanolamine (but not serine, glycine, dimethylglycine, methylamine, hydroxylamine or beta-hydroxyethylhydrazine) to the medium also resulted in appreciable increases in the colony radial growth rates of A3/5 (increased by about 130% for choline) and C106 (increased by about 25% for choline). No significant difference was observed between the phospholipid compositions of the two strains, and the addition of 100 microM-choline to the medium had no significant effect on the phospholipid composition of either strain.     

Effects of exogenous phospholipids on lipid composition and sporulation by three strains of Lagenidium giganteum.

The California (LGCA) and Butte Sink (LGBS) strains of the sterol auxotrophic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) enter the sexual cycle on media supplemented with sterols. A third isolate of this mosquito pathogen, the North Carolina strain (LGNC), requires sterols plus phospholipids to produce oospores in vitro. Enrichment of the polar and neutral lipid fractions of the LGCA and LGBS strains with unsaturated fatty acids promoted oospore induction, and increased oospore viability. With the exception of the LGCA strain, there was no consistent relationship between phospholipid supplementation in growth media and mycelial phospholipid content.     

Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains

The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation of fluconazole thereby providing a mechanism for azole resistance.     

Modification of the fatty acid composition of phospholipid in measles virus-persistently infected cells

The composition of phospholipid was studied in BGM cells uninfected, persistently infected or lytically infected with measles virus, strain Hallé. In persistently infected cells, phosphatidylcholine palmitic acid content, and phosphatidylethanolamine palmitic acid and arachidonic acid contents were significantly increased. Lytically infected cells had a similar phospholipid fatty acid composition to the uninfected. Phosphatide composition showed minor modifications, but the content of total choline-derivative phospholipids was unchanged in either type of infection.     

Circadian rhythms in Neurospora crassa: a clock mutant, prd-1, is altered in membrane fatty acid composition

The fatty acid compositions of the phospholipids of Neurospora crassa mutants with altered periods were determined to test the possibility that some of these mutants might have altered membrane composition. In liquid shaker culture in constant light the bd (band) strain, which has a normal period (21.6 h), exhibited a growth-dependent increase in linoleic acid content and a decrease in linolenic acid content during early log phase growth. By late log phase, fatty acid composition was essentially constant. The phospholipid fatty acid compositions of bd strains containing mutations at the frq (frequency) and chr (chrono) loci were indistinguishable from that of the bd strain under the conditions used. However, a bd strain containing a mutation at the prd-1 (period) locus, as well as prd-1 segregants from a cross of this strain to a bd strain, had altered patterns of growth-dependent fatty acid composition; linoleic and linolenic acid contents changed more slow than in the bd strain and continued to change throughout growth. In addition, the fatty acid composition of a bd prd-1 strain on solid medium differed from that of the bd strain. It is proposed that the prd-1 mutation leads to altered membrane homeostasis, which in turn affects circadian rhythmicity because some or all components of the rhythm-generating system are membrane-localized.     

Lipid Content of Antibiotic-Resistant and -Sensitive Strains of Serratia marcescens

The lipid content of antibiotic-resistant, nonpigmented strain (Bizio) and antibiotic-sensitive, pigmented strain (08) of Serratia marcescens was studied. The resistant strain contains at least three times more total extractable lipid and phospholipid than the sensitive strain. Lysophosphatidylethanolamine, phosphatidylserine, lecithin, phosphatidylglycerol, phosphatidylethanolamine, and polyglycerolphosphatide were identified in the phospholipid fractions of both strains.     

Glycerophospholipid variation in choline and inositol auxotrophs of Neurospora crassa. Internal compensation among zwitterionic and anionic species.

The glycerophospholipids of cultures of Neurospora crassa were extracted, deacylated, and analyzed. In addition to a wild-type strain, several auxotrophic mutant strains were examined: chol-1 (defective S-adenosylmethionine: phosphatidylethanolamine methyltransferase), chol-2 (defective S-adenosyl methionine:phosphatidylmonomethylethanolamine (dimethylethanolamine) methyltransferase), and inos (defective myoinositol-1-phosphate phosphatase). In addition, a double mutant strain, chol-1;chol-2, was constructed. Cultures of the mutant strains grown with concentrations of supplement(s) just adequate to support growth had bizarre phospholipid compositions. By appropriate choice of mutant and supplement(s), it was possible to vary the relative level of every phospholipid of the organism, with the exception of cardiolipin. The maximum ranges encountered for the zwitterionic species, expressed as per cent of total phospholipid phosphorus, were lecithin (0.9 to 53.1%), phosphatidyldimethylethanolamine (0.0 to 55.5%), phosphatidylmonomethylethanolamine (0.0 to 53.9%), and phosphatidylethanolamine (9.8 to 43.3%). For the anionic species, the ranges were phosphatidylserine (1.7 to 10.4%) and phosphatidylinositol (3.6 to 25.1%). Despite the wide variation of the relative proportions of the individual phospholipid species, five quantities remained constant: the cardiolipin content, the total phospholipid content, the total content of the zwitterionic species, the total content of the anionic species, and the ratio of the zwitterionic to anionic totals. The data suggest the existence of an internal compensation mechanism, the net effect of which is maintenance of a fairly constant contribution by the phospholipid components to the over-all membrane charge.